Uropathogens and their antibiotic susceptibility patterns among diabetic patients at st. john of god hospital, duayaw nkwanta, Ghana: a cross‐sectional study

Abstract Background Uropathogens are microorganisms that cause urinary tract infections (UTIs). Owing to higher blood glucose levels and compromised immune functions, treatment of uropathogens in diabetic patients is a challenge. Aim This study aims to assess the prevalence of uropathogens and their antibiotic susceptibility among diabetic patients at St. John of God Hospital at Duayaw Nkwanta (SJGHDN) in the Ahafo region of Ghana. Methods The cross‐sectional study recruited 175 diabetic patients at SJGHDN between August and September 2023. Questionnaires were used to collect patients’ background information. Fasting Blood Sugar (FBS) was assessed by using a glucometer. Urine samples were examined for the presence of uropathogens. A sterile inoculating loop with a calibrated volume of 2 µl was used for plating. Each colony equals 500 CFU/mL. Significant uropathogen was determined by multiplying the counted colonies by 500 to obtain CFU/mL. Positive uropathogen was defined as CFU ≥ 105/mL. Significant uropathogen was defined as ≥200 colonies per sample. The disc diffusion method was used to determine antibiotic susceptibility. Results Out of the 175 patients, 19.4% expressed various uropathogens with Escherichia coli being the predominant. Suboptimal glucose level was the most significant risk factor (p = 0.038). Glucosuria (p = 0.036), hazy urine (p = 0.028), positive leukocyte esterase (p = 0.001), and pus cells in urine sediment (p = 0.020) were significant indicators of uropathogen occurrence. Klebsiella pneumonia and Proteus mirabilis were resistant to ≥4 antibiotics. Amikacin, nitrofurantoin, levofloxacin, ciprofloxacin, and ceftriaxone demonstrated efficacy against the isolates. Conclusion This study underscores the notable prevalence of uropathogens in diabetic patients and the alarming levels of antibiotic resistance observed. The results highlight the critical need for vigilant monitoring and customized treatment approaches, particularly for diabetic patients exhibiting risk factors such as elevated urine glucose levels, cloudy urine, and presence of leukocyte esterase and pus cells in urine sediment. The significant resistance to frequently used antibiotics like co‐trimoxazole and tetracycline points to the necessity of routine susceptibility testing and the use of alternative antibiotics for effective treatment. These findings can assist healthcare providers in more effectively managing and preventing UTIs in diabetic populations.

and the rate of occurrence could be as high as 23.4% to 37% in women.
Coincidentally, the studies identified Escherichia coli (E.coli), Enterococcus species (Enterococcus.spp), Klebsiella pneumoniae (K.pneumoniae), Proteus mirabilis (P.mirabilis), Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S. aureus), Coagulase-negative staphylococci (CoNS), and Enterobacter species (Enterobacter.4][5][6][7] Though diabetes, uropathogens, and rUTIs are global health threats, low-income countries (LICs) are affected most due to socio-cultural and socioeconomic diversities, as well as poor diagnosis and treatment as a result of resource limitations. 8,9However, the magnitude of occurrence of uropathogens and rUTIs among T2DM patients in the LICs is underexplored. 10In Ghana, the prevalences of T2DM and uropathogens in T2DM patients are reported to be 3.95% and 9.2% respectively. 1,11Due to cultural and economic diversities, managing this condition is more challenging.For instance, some patients in remote areas do not go for regular check-ups and treatments because they cannot afford transportation expenses.Also, poor hygienic practices such as drinking contaminated water, eating contaminated food, and sharing public toilet facilities that are sometimes not sanitized, could enhance the spread of resistant uropathogens.
Though the intersection of T2DM and the occurrence of uropathogens and rUTIs in Ghana has been reported, there is still a knowledge gap on the contribution of the health facilities, hygienic practices, and healthseeking behavior of T2DM patients to the occurrence of uropathogens and rUTIs.Also, there is limited data regarding the reliable predictors of the occurrence of uropathogens and rUTIs in patients.Given the above, this study aims to explore the prevalence of UTI and the antimicrobial resistance patterns among diabetic patients in Ghana.It will also examine whether health-seeking behaviour and sanitation standards affect the study cohort's spread of uropathogens and rUTIs.The novelty of this research lies in its comprehensive approach to unraveling the relationship between uropathogens, rUTIs, and sanitation standards among T2DM patients in the study setting, which has been underexplored in the existing literature.The findings of this study have the potential to inform targeted interventions, guide policy formulation, and ultimately improve the quality of life for diabetic patients in Ghana.Through this research, we aim to contribute to the global discourse on T2DM and UTIs, providing valuable insights that can be applied to similar contexts worldwide.

| Study design
A cross-sectional study was conducted between August and September 2023 on diabetic patients receiving treatment at SJGHDN in the Ahafo region of Ghana.Participants were confirmed diabetes type 2 clients at the Hospital.Diagnoses were made based on fasting glucose levels of 126 mg/dL or higher and HbA1c levels of 6.5% or higher. 12,13These patients have unique ID cards.A convenient sampling technique was used to recruit study participants.

| Inclusion criteria
The study exclusively enrolled individuals ≥18 years old with confirmed type 2 DMs patients attending the diabetic review clinic between August and September 2023.

| Sample size calculation
The minimum sample size for the study was determined by using the For the study, a minimum sample size of 129 diabetics was significant.A total of 175 diabetics meeting the inclusion criteria were enrolled to increase the study's statistical power.

| Exclusion criteria
Individuals with DM who were presently undergoing antibiotic treatment for any medical condition were excluded from this study to prevent potential confounding factors associated with ongoing antibiotic therapy.to produce clean, midstream urine.For the specimen in container A, the semi-quantitative assessment of urine leucocytes, glucose, protein, nitrites, pH, and specific gravity was conducted using Mission ® Combi-Ten urine biochemistry strips.Subsequently, the urine sample was centrifuged at 1500 revolutions per minute (rpm) for 5 min. 14e supernatant was meticulously discarded, and a single droplet of the sediment was delicately placed onto a standard microscope slide and covered with a 22 × 22 mm coverslip.The examination of the urine specimens for the presence of pus cells, red blood cells, epithelial cells, and Trichomonas vaginalis was performed using the low objective lens (x40) of the Olympus ® CX 21 microscope and recorded.

| Urine culture
Cysteine lactose electrolyte-deficient (CLED) agar was used as a culture medium based on its ability to demonstrate morphologic differentiation of colonies and prevent Proteus species.from swarming.As a nutrient agar, it supports the growth of all uropathogens and has demonstrated 100% agreement with other standard agars used in isolating uropathogens.CLED has widely been used in other works of similar nature. 15,16Briefly, urine specimens in container B were plated on the CLED agar plate.A sterile inoculating loop with a calibrated volume of 1/500 ml (2 µl) was used. 14A loop full of urine specimens was streaked on a CLED agar plate using the quarter plating technique.Urine specimens with a cloudy appearance were limited to three specimens per agar plate.The plate was incubated at 37°C for 24 h.After the incubation, agar plates were observed for growth, and colonies were observed for their lactose fermentation pattern and colony morphology.The colonies were counted, and significant uropathogen was determined by multiplying the counted colonies by 500 to obtain colony-forming units per ml (CFUs/ml).The threshold for the definition of a significant uropathogen is 10  States of America).For categorical data, numbers and proportions for each category were presented and the mean was used to represent the continuous age of participants.The chi-square test statistic [18][19][20] was used to assess the significant association between uropathogens presence and patients' characteristics.2][23] Statistical significance was defined as p < 0.05.

| Lifestyle and clinical features of the study participants
The results in

| Drug susceptibility pattern of uropathogens
The susceptibility patterns of uropathogens are illustrated in Figure 1.
The The table shows how socio-demographic, lifestyle, and clinical variables could be used to predict the presence of uropathogens.Suboptimal glucose levels (6.6-10 mmol/L), urine glucose of +3, hazy urine, trace and +2 levels of urine leukocytes, and pus cells of +3 and +5 were found to be significant predictors of the presence of uropathogens.
5][26] Contrary to the above reports, studies conducted in the USA, Romania, Italy, and Canada found lower UTI prevalent rates of 8.2%, 12.0%, 14.9%, and 7.9% respectively. 27,28ese differences in prevalent rates may be due to differences in climatic and environmental factors that could influence the distribution of uropathogens.Also, maintaining optimal glucose levels is expensive for diabetic patients in LICs.Therefore, failure to maintain optimal glucose levels provides a suitable environment for microbial invasion, which may account for the higher prevalent rates in LICs. 24 is also worth mentioning that prevalent rates of different geographical areas of the same country may widely differ.For instance, while our current study found uropathogen occurrence in the Bono region of Ghana to be 19.4%, a similar study in Accra, Ghana's capital, reported a lower prevalence of 9.2%. 11This variation in prevalence within the same country may be due to population dynamics and resource disparities between the two areas.For instance, there are many ultra-modern diabetic clinics and affluent people in Accra than in the remote areas like the Bono region where our study was conducted.These discrepancies have also been reported in Ethiopia and Uganda. 29,30In terms of gender, 88.2% of the T2DM patients who tested positive for uropathogens were women.This finding is similar to the studies conducted in Ghana, Pakistan, Uganda, Iran, and Ethiopia, where rates of bacteriuria in females were respectively found to be 86.7%,0][31][32][33] Women's anatomical and physiological predisposition may account for this higher incidence of bacteriuria. 26Though studies conducted in Uganda and Germany observed a positive correlation between the age of T2DM patients and the occurrence of uropathogen, 26,34 our study did not find any significant association between age and bacteriuria.Our finding is consistent with the reports by Al-Rubeaan et al., 35 Hamdan et al., 24 and Mama et al. 29 Also, the occurrence of uropathogen in T2DM patients was significantly associated with neither the duration of diabetes, the type of toilet facility used nor the presence of co-morbidities identified in this study.This observation supports the reports of similar studies conducted in Saudi Arabia and Ethiopia where the above factors did not have any association with the occurrence of uropathogens. 35,368][39] It is therefore imperative for T2DM patients to maintain optimal glucose levels to prevent the incidence of uropathogens and UTIs.
Out of the 34 culture-positive isolates in this study, E. coli was the predominant (50%) followed by Gram-positive Enterococci spp.
Furthermore, our study identified amikacin, nitrofurantoin, levofloxacin, and ceftriaxone as first-line drugs for treating UTIs caused by gram-negative rods.Interestingly, studies conducted in Ghana, Ethiopia, and India also found these antibiotics as the best drugs for treating UTIs caused by gram-negative bacteria. 7,15,29

STUDY LIMITATION
This study involved only 175 T2DM patients due to time and resource constraints.Since this figure represents a small fraction of T2DM patients seeking healthcare at SJGHDN per year, the study can be replicated by extending the duration and the participants so that the results can reflect the entire T2DM population of the Bono region of Ghana.
Cochran formula n = z 2 * p (1-p)/m 2 , where n = minimum sample size, z = standard normal variance = 1.96 to obtain a power of 95% confidence interval and a type 1 error probability of 5%.P = average prevalence of UTI was 9.2% according to Forson et al. (2021).11

From a 24 -
h pure culture, 4-6 bacterial colonies were gathered using a sterile loop and transferred to a tube containing 2 ml of sterile normal saline.The mixture was gently agitated to achieve a uniform suspension, whose turbidity matched a 0.5% McFarland standard.A sterile cotton swab was dipped into the saline suspension and pressed against the side of the tube before being used to streak and cover the entire surface of the plate.Axiom ® multidiscs for urinary isolates were evenly placed on the inoculated agar plates.The antibiotics used in the susceptibility tests were: ampicillin/sulbactam (20 µg), cotrimoxazole (25 µg), amikacin (30 µg), chloramphenicol (30 µg), cephalexin (30 µg), tetracycline (30 µg), ciprofloxacin (5 µg), nitrofurantion (300 µg), ceftriaxone (30 µg), levofloxacin (5 µg), norfloxacin (10 µg), and ofloxacin (5 µg).The plates were incubated in air at 37°C for 24 h.The diameter of the zone of inhibition was measured after 24 h of incubation.The zone of inhibition was classified into sensitive, intermediate sensitive, and resistant.2.7 | Data analysisData was entered into Microsoft Excel and analyzed using the Statistical Package for Social Sciences version 26.0 (Chicago, United

F I G U R E 1
Antibiogram susceptibility pattern for Gram-negative and Gram-positive bacteria.K. pneumoniae was 100% resistant to levofloxacin, tetracycline, norfloxacin, ciprofloxacin, and ofloxacin.P. mirabilis was 100% resistant to tetracycline, norfloxacin, and ampicillin/ sulbactam.S. aureus was 100% resistant to norfloxacin and chloramphenicol.Saudi Arabia, Bangladesh, Portugal, USA, Tanzania, Nepal, Pakistan, and Cameroun.11,[42][43][44][45][46][47][48][49]Gram-positive isolates, including Staphylococcus aureus, were 100% susceptible to amikacin but 100% resistant to ampicillin+sulbactam and chloramphenicol.This resistant pattern is consistent with the findings ofWoldemariam et al. (2019) and Bessong et al. (2013).7,49The prescription of antibiotics for treating UTIs in T2DM patients receiving healthcare at SJGHDN must follow the Laboratory description of the causative organism to ensure a successful treatment outcome.5 | CONCLUSIONThis study highlights the significant prevalence of uropathogens among diabetic patients and the concerning levels of antibiotic resistance.The findings underscore the need for careful monitoring and tailored treatment strategies for diabetic patients, especially those with risk factors such as high urine glucose levels, hazy urine appearance, and the presence of leukocytes esterase and pus cells in urine sediment.The high resistance rates to commonly used antibiotics like co-trimoxazole and tetracycline emphasize the importance of regular susceptibility testing and the consideration of alternative antibiotics for effective treatment.These insights can guide healthcare providers in managing and preventing urinary tract infections in diabetic populations more effectively.

Table 1
presents the percentage distribution of the sociodemographic characteristics of the study participants.Out of the 175 participants, 144 (82.3%) were females and 31 (17.7%) were males.The mean age of the participants was 60.89 ± 12.81 years.The majority of diabetes patients, 96 out of 175 (54.9%), are between the ages of 50 and 69.

Table 2
Socio-demographic variables of participants.Lifestyle and Clinical Factors.
overall percentages of drug-resistant uropathogens were as follows: E. coli at 18.18% (2 out of 11), P. aeruginosa at 60% (3 out of 5), Enterobacter spp. at 36.36% (4 out of 11), K. pneumoniae at 50.00% (5 out of 10), P. mirabilis at 27.27% (3 out of 11), Enterococcus spp. at 57.14% (4 out of 7), CoNS at 50.00% (4 out of 8), and S. aureus at 87.50% (7 out of 8).The Gram-negative isolates showed 100% susceptibility to amikacin.Specifically, E. coli exhibited high susceptibility to amikacin This study aimed to ascertain the prevalence of uropathogens and their corresponding antibiotic susceptibility profiles in diabetic patients receiving treatment at St. John of God Hospital, Duayaw Nkwanta, in the Bono region of Ghana.The prevalence of uropathgens among the cohort was 19.4%.This is comparable to the studies T A B L E 3 Summary of Uropathogens Isolate.